Evaluation of tumor mutation burden (TMB) in tumor (tDNA) and plasma cell free DNA (cfDNA) in patients (pts) with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) treated with a combination of cetuximab and nivolumab (C+N).
Authors: Christine H. Chung, Graydon Moorhead, Nicholas Haradhvala, Laxmi Parida, Jiannong Li, Robbert Slebos, Xiaofei Song, Ritu Chaudhary, Maria Poole, Robin Park, Kedar Kirtane, Conor Ernst Steuer, Nabil F. Saba, Priyanka Bhateja, James William Rocco, Marcelo Raul Bonomi, Brian Danysh, Gad Getz, Aik-choon Tan
Journal: Journal of Clinical Oncology (2023)

6053 Background: High TMB is associated with immune checkpoint inhibitor (ICI) response in many solid tumors. However, obtaining sufficient tumor specimens for testing in a routine clinical setting could be a challenge. We evaluated TMB from whole-exome sequencing (WES) of cfDNA as a surrogate for tDNA TMB in R/M HNSCC pts treated with C+N. Methods: Archived formalin fixed paraffin embedded tumors, serially collected plasma (pre-treatment, on-treatment, and time of disease progression/end of treatment), peripheral mononuclear blood cells, and clinical data were obtained from R/M HNSCC pts treated with C+N from a completed clinical trial (NCT03370276). Nucleic acids were extracted using AllPrep DNA/RNA Mini Kit (Qiagen). Libraries were constructed using KAPA HyperPrep Kit with Library Amplification (KK8504) and IDT’s duplex UMI adapters. Hybridization and capture were performed using IDT’s XGen hybridization kit. Whole exome sequencing was performed using Illumina NovaSeq S4 flowcells (paired 151bp runs). Data preprocessing including point mutation calling, filtering, CNV calling, and purity and ploidy estimation were performed using the Getz Lab CGA WES Characterization pipeline. TMB for each sample was calculated based on the number of mutations with cancer cell fraction &gt; 0.75 as determined by ABSOLUTE and normalized to the 35Mb targeted for exome capture. Results: A total of 88 tDNA, 226 cfDNA, and 30 normal DNA (nDNA) from 82 pts were sequenced. To date, sequencing data from 13 matched tDNA/cfDNA/nDNA samples and 34 matched cfDNA/nDNA samples with sufficient tumor purity were available for analyses. The median TMB was 2.4 mut/Mb (range 0.46-17.5) in tDNA and 1.8 mut/Mb (range 1.1-12.6) in cfDNA. There was a high correlation between tDNA/cfDNA pairs in TMB (r = 0.826, p = 1.5x10 -16 , Pearson correlation). The median TMBs in tDNA and cfDNA obtained at different time points or treatment course did not differ significantly in 22 patients with at least two cfDNA timepoints (p = 0.26 paired t-test of log-transformed TMB). There was no difference between responder (R) vs. non-responder (NR) in cfDNA TMBs (two-sample t-test p = 0.7 between 2 R and 12 NR) and tDNA TMBs (two-sample t-test p = 0.25 between 6 R and 21 NR). However, tDNA TMB was higher in R compared NR in a subset of pts who did not have prior treatment with ICI (two-sample t-test p = 0.05 between 5 R and 2 NR). Conclusions: TMB in tDNA and cfDNA is highly correlated and suggests the possible use as a surrogate marker for tTMB. High TMB in cfDNA derived from ICI-naïve patients appears to have a prognostic value. Further evaluation is warranted to determine the role of cfDNA TMB as a biomarker of ICI response.